Behnaz Ahrabi
1,2 
, Hojjat Allah Abbaszadeh
3,2,1* , Abbas Piryaei
4,1, Faezeh Shekari
5,6, Navid Ahmady Roozbahany
7, Mahya Rouhollahi
5, Forough Azam Sayahpour
5, Mahnaz Ahrabi
8, Hadi Azimi
9, Reza Moghadasali
5* 1 Department of Biology and Anatomical Sciences, school of medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran
2 Laser Applications in Medical Sciences Research Center, Shahid Beheshti University of Medical Sciences, Tehran, Iran
3 Hearing Disorders Research Center, Loghman Hakim Hospital, Shahid Beheshti University of Medical Sciences, Tehran, Iran
4 Urogenital Stem Cell Research Center, Shahid Beheshti University of Medical Sciences, Tehran, Iran
5 Department of Stem Cells and Developmental Biology, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, Iran
6 Advanced Therapy Medicinal Product Technology Development Center, Royan Institute for Stem Cell Biology and Technology, Academic Center for Education, Culture and Research, Tehran, Iran
7 Private Practice, Bradford, ON, Canada
8 Department of Endodontics, Dental Faculty, Tabriz University of Medical Sciences, Tabriz, Iran
9 Department of English Language Teaching, School of Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran
Abstract
Introduction: Chronic and progressive damage to the kidney by inflammatory processes, may lead to an increase in the extracellular matrix production, a condition known as renal fibrosis. The current study aims to evaluate if the extracellular vesicles (EVs) derived from autophagic adipose-derived mesenchymal stem cells (ADMSCs) can reduce the inflammation and extracellular matrix accumulation in damaged kidney tissue.
Methods: Autophagy was induced in ADMSCs using 2µM concentration curcumin and was confirmed by evaluating LC3B, ATG7, and Beclin1 using real-time polymerase chain reaction (PCR) and Western blot. An in vitro renal fibrotic model was established in HEK-293 cells exposed to H2O2 (0.8mM) for 24 and 72 hours. The fibrotic model was confirmed through evaluation of collagen I, transforming growth factor-beta 1 (TGF-β1), E-cadherin, and vimentin genes expression using real-time PCR, collagen I protein by ELISA. After induction of fibrosis for 24 and 72 hours, the HEK cells were treated with NEVs (non-autophagy EVs) (50µM) or AEVs (autophagy EVs) (50µM) at 48, 96, and 124 hours, and then the samples were collected at 72 and 148 hours. Expression of collagen I, TGF-β1, E-cadherin, and vimentin Genes was evaluated via RT-PCR, and protein levels of IL1, TNF-α, IL4, IL10 using ELISA.
Results: Induction of autophagy using curcumin (2µM) for 24 hours significantly increased LC3B, Beclin1, and ATG7 in the ADMSCs. Upregulation in anti-fibrotic (E-cadherin) and anti-inflammatory (IL4, IL10) gene expression was significantly different in the fibrotic model treated by AEVs compared to NEVs. Also, the downregulation of fibrotic (TGF-β1, vimentin, collagen I) and pro-inflammatory (IL1, TNFα) gene expression was significantly different in AEVs compared with those treated by NEVs.
Conclusion: Our findings suggest that AEVs can be considered as a therapeutic modality for renal fibrosis in the future.